Arboviruses cause a significant world-wide health burden, with well over 100 million people becoming infected each year with viruses such as dengue, West Nile, yellow fever, and chikungunya, among others. Arboviruses are transmitted by arthropod vectors such as mosquitoes that become infected after ingestion of a viremic blood meal from a vertebrate host. Transmission to a new host requires that the arbovirus replicate in the midgut cells of the vector and then spread to secondary tissues eventually reaching the salivary glands. Once the latter are infected, the arthropod is able to transmit the virus to a new host. A long standing question in the field of vector biology is how arboviruses escape from the midgut, bypassing barriers such as basal laminae as well as host immune mechanisms. In some cases, arboviruses are able to infect and replicate in midgut epithelium but are not able to disseminate to other organs. The existence of this so-called midgut escape barrier implies that virus midgut escape is an active process. However, the mechanisms involved in midgut escape by arboviruses are almost completely unknown. This proposal addresses the signaling mechanisms used by the mosquito-borne viruses dengue, chikungunya, and Sindbis viruses to escape the midgut. Previous work by the investigators has defined a novel mechanism used by baculovirus to escape the midgut of their insect host. Baculoviruses use an elegant mechanism that signals a stepwise cascade of protease activation, wherein matrix metalloproteases become activated and in turn activate effector caspases, which directly cleave components of the basal lamina. This leads to remodeling of the basal lamina which lines tracheal cells associated with the midgut and culminates in the establishment of efficient systemic infections. The hypothesis underlying this proposal is that mosquito-borne arboviruses utilize this same pathway for midgut escape, and preliminary results support this hypothesis. Specifically, (1) it will be determined whether the mechanisms used by baculoviruses to remodel the midgut barrier are also utilized by arboviruses, including activation of matrix metalloproteases and caspases; (2) the contribution of candidate genes involved in midgut escape will be evaluated by RNA interference, arbovirus transducing systems, and transgenic Aedes aegypti mosquitoes by silencing or overexpressing target genes; and (3) the contribution of apoptosis and key apoptotic regulatory genes to arbovirus midgut escape will be tested. The results of these studies will contribute important new information to the understanding of arbovirus-vector interactions and potentially lead to new strategies for control of arbovirus transmission in the field.